Mixture of a positive and negative contrast agent for magnetic resonance imaging

ABSTRACT

There is provided a method of generating enhanced images of the human or non-human animal body, for example for use in medical diagnosis, which involves administering to the body a positive MRI contrast agent which is body tissue- or body duct-specific following the particular mode of administration and a negative MRI contrast agent which preferably also is body tissue- or body duct-specific. Thereafter a magnetic resonance image is generated of a part of the body containing the negative and positive contrast agents or their paramagnetic, ferromagnetic or superparamagnetic biodegradation products. Contrast media suitable for use in this new image generating method are also provided.

This invention relates to improvements in and relating to magneticresonance imaging (MRI) and more particularly to a novel method ofenhancing contrast in MRI and to contrast media therefor.

In MRI, the contrast in the images generated may be enhanced byintroducing into the zone being imaged an agent, generally referred toas a contrast agent, which affects the spin reequilibrationcharacteristics of the nuclei (the "imaging nuclei" which generally areprotons and more especially water protons) which are responsible for theresonance signals from which the images are generated. The enchancedcontrast obtained with the use of contrast agents enables particularorgans or tissues to be visualized more clearly by increasing or bydecreasing the signal level of the particular organ or tissue relativeto that of its surroundings. Contrast agents raising the signal level ofthe target site relative to that of its surroundings are termed"positive" contrast agents whilst those lowering the signal levelrelative to surroundings are termed "negative" contrast agents.

The majority of materials now being proposed as MRI contrast mediaachieve a contrast effect because they contain paramagnetic,superparamagnetic or ferromagnetic species.

For ferromagnetic and superparamagnetic contrast agents, which arenegative MRI contrast agents, the enhanced image contrast derivesprimarily from the reduction in the spin reequilibration coefficientknown as T₂ or as the spin-spin relaxation time, a reduction arisingfrom the effect on the imaging nuclei of the fields generated by theferromagnetic or superparamagentic particles.

Paramagnetic contrast agents on the other hand may be either positive ornegative MRI contrast agents. The effect of paramagnetic substances onmagnetic resonance signal intensities is dependent on many factors, themost important of which are the concentration of the paramagneticsubstance at the imaged site, the nature of the paramagnetic substanceitself and the pulse sequence and magnetic field strength used in theimaging routine. Generally, however, paramagnetic contrast agents arepositive MRI contrast agents at low concentrations where their T₁lowering effect dominates and negative MRI contrast agents at higherconcentrations where their T₂ lowering effect is dominant. In eitherevent, the relaxation time reduction results from the effect on theimaging nuclei of the magnetic fields generated by the paramagneticcentres.

The use of paramagnetic, ferromagnetic and superparamagnetic materialsas MRI contrast agents has been widely advocated and broad ranges ofsuitable materials have been suggested in the literature.

Thus, for example Lauterbur and others have suggested the use ofmanganese salts and other paramagnetic inorganic salts and complexes(see Lauterbur et al. in "Frontiers of Biological Energetics", volume 1,pages 752-759, Academic Press (1978), Lauterbur in Phil. Trans. R. Soc.Lond. B289: 483-487 (1980) and Doyle et al. in J. Comput. Assist.Tomogr. 5(2): 295-296 (1981)), Runge et al. have suggested the use ofparticulate gadolinium oxalate (see for example U.S. Pat. No. 4,615,879and Radiology 147(3): 789-791(1983)), Schering AG have suggested the useof paramagnetic metal chelates, for example of aminopolycarboxylic acidssuch as nitrilotriacetic acid (NTA),N,N,N',N'-ethylenediaminetetraacetic acid (EDTA),N-hydroxyethyl-N,N',N'-ethylenediaminetriacetic acid (HEDTA),N,N,N',-N",N"-diethylenetriaminepentaacetic acid (DTPA), and1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) (see for exampleEP-A-71564, EP-A-130934, DE-A-3401052 and U.S. Pat. No. 4,639,365), andNycomed AS have suggested the use of paramagnetic metal chelates ofiminodiacetic acids (see EP-A-165728). Besides paramagnetic metals,paramagnetic stable free radicals have also been suggested for use aspositive MRI contrast agents (see for example EP-A-133674).

Other paramagnetic MRI contrast agents are suggested or reviewed in, forexample, EP-A-136812, EP-A-185899, EP-A-186947, EP-A-292689,EP-A-230893, EP-A-232751, EP-A-255471, WO85/05554, WO86/01112,WO87/01594, WO87/02893, U.S. Pat. No. 4,639,365, U.S. Pat. No.4,687,659, U.S. Pat. No. 4,687,658, AJR 141: 1209-1215 (1983), Sem.Nucl. Med. 13: 364 (1983), Radiology 147: 781 (1983), J. Nucl. Med. 25:506 (1984), WO89/00557 and International Patent Application No.PCT/EP89/00078.

Ferromagnetic (a term used herein to cover both ferrimagnetic andferromagnetic materials) and superparamagnetic MRI contrast agents, forexample sub-domain sized magnetic iron oxide particles either free orenclosed within or bound to a particle of a non-magnetic matrix materialsuch as a polysaccharide, are disclosed by Schroder and Salford inWO85/02772, by Nycomed AS in WO85/04330, by Widder in U.S. Pat. No.4675173, by Schering AG in DE-A-3443252 and by Advanced Magnetics Inc inWO88/00060.

Intravenous administration, at separate times, of the positive contrastagent Gd DTPA-dimeglumine (which following such administration rapidlydistributes extracellularly) and of superparamagnetic ferrite particleswas proposed by Weissleder et al. in AJR 150: 561-566 (1988) for imagingof liver cancers and by Carvlin et al. Society for Magnetic ResonanceImaging, 5th Annual Meeting, San Antonio, 1987, for studying renalbloodflow. Carvlin and Weissleder's work on this topic is reportedfurther in Proc. SPIE-Int.Soc.Opt.Eng. (1988) 914 Medical Imaging II,Pages 10-19 and AJR 150 115-120 (1988) respectively.

The present invention arises from the recognition that tissue or organvisualization in MRI may surprisingly be particularly enhanced byadministration of tissue- or body duct-specific positive and negativecontrast agents, despite their diametrically opposed contrast effects.

Thus, in one aspect, the present invention provides the use of bodytissue- or body duct-specific positive and negative MRI contrast agentse.g. a physiologically acceptable paramagnetic substance and aphysiologically acceptable ferromagnetic or superparamagnetic substance,for the manufacture of a contrast medium for use in a method ofdiagnosis practised on the human or non-human animal body, a whichmethod comprises administering to said body a body tissue- or bodyduct-specific negative MRI contrast agent and a body tissue- or bodyduct-specific positive MRI contrast agent and generating a magneticresonance image of a part of said body containing said negative andpositive contrast agents or paramagnetic, ferromagnetic orsuperparamagnetic biodegradation products thereof.

In a further aspect, the present invention provides the use of a bodytissue- or body duct specific positive MRI contrast agent, e.g. aphysiologically acceptable paramagnetic substance, for the manufactureof a contrast medium for use in a method of diagnosis practised on thehuman or non-human animal body, which method comprises administering tosaid body a body tissue- or body duct-specific negative MRI contrastagent and a said positive MRI contrast agent and generating a magneticresonance image of a part of said body containing said negative andpositive contrast agents or paramagnetic, ferromagnetic orsuperparamagnetic biodegradation products thereof.

In a yet further aspect, the present invention provides the use of abody tissue- or body duct-specific negative MRI contrast agent, e.g. aphysiologically acceptable ferromagnetic or superparamagnetic substance,for the manufacture of a contrast medium for use in a method ofdiagnosis practised on the human or non-human animal body, which methodcomprises administering to said body a said negative MRI contrast agentand a body tissue- or body duct-specific positive MRI contrast agent andgenerating a magnetic resonance image of a part of said body containingsaid negative and positive contrast agents or paramagnetic,ferromagnetic or superparamagnetic biodegradation products thereof.

In a still further aspect, the present invention provides a method ofdiagnosis practised on the human or non-human animal body, which methodcomprises administering to said body an effective amount of a bodytissue- or body duct-specific negative MRI contrast agent and aneffective amount of a body tissue- or body duct-specific positive MRIcontrast agent, e.g. a physiologically acceptable paramagnetic substanceand a physiologically acceptable ferromagnetic or superparamagneticsubstance, and generating a magnetic resonance image of a part of saidbody containing said paramagnetic substance and said ferromagnetic orsuperparamagnetic substance or paramagnetic, ferromagnetic orsuperparamagnetic biodegradation products thereof.

In a yet still further aspect the invention provides a method ofgenerating images of the human or non-human animal body, which methodcomprises administering to said body a body tissue- or bodyduct-specific negative MRI contrast agent and a body tissue- or bodyduct-specific positive MRI contrast agent, e.g. a physiologicallyacceptable paramagnetic substance and a physiologically acceptableferromagnetic or superparamagnetic substance, and generating a magneticresonance image of a part of said body containing said negative andpositive agents or paramagnetic, ferromagnetic or superparamagneticbiodegradation products thereof.

By tissue or duct specific it is meant that the agent and administrationroute are such that following administration the agent does notdistribute widely but instead is substantially maintained within a bodyduct or cavity throughout the period necessary for image generation oris caused to concentrate at a particular body tissue or organ due to theinteraction of the body and the agent. Thus particularly preferably thetissue or duct specific agent may be a blood pool agent administeredinto and then retained within the cardiovascular system (unlike theextracellularly distributing GdDTPA which after iv administrationrapidly distributes into a body volume about five times larger than thatof the circulatory system). In another preferred embodiment, the tissueor duct specific agent is a tissue targetting agent, such as thehepatobiliary positive contrast agents of EP-A-165728 or thereticuloendothelial system targetting negative contrast agents ofWO85/04330. However, for the purposes of the methods according to theinvention extracellularly-distributing paramagnetic metal-containingpositive contrast agents, such as Gd DTPA, Gd DOTA and Gd DTPA-BMA (thegadolinium chelate of the bismethylamide of DTPA), may be used accordingto the present invention for administration into body cavities or tractshaving externally voiding ducts, e.g. for oral administration into thegastrointestinal tract, since they are not absorbed into the body fromsuch cavities or tracts and using such administration routes can beconsidered to be "duct specific".

In accordance with the invention, the positive and negative contrastagents are duct or tissue specific. Particularly preferably thespecificity of the agents should be such that at the sites of particularinterest for imaging the two should distribute within different bodyvolumes Where the agents are administered via the same administrationroute, e.g. into the cardiovascular system, this will mean thatoperation of the body organ or tissue to separate the agents will resultin enhanced contrast of the agents. Alternatively, however, the agentscan be administered via different routes and caused to approach eachother or to merge in the body region being imaged.

In one particularly preferred embodiment of the method and use of theinvention, the positive and negative contrast agents are administeredtogether as a single composition and in a still further aspect theinvention thus provides a magnetic resonance imaging contrast mediumcomposition comprising at least one physiologically acceptable bodytissue- or body duct-specific negative MRI contrast agent together withat least one physiologically acceptable body tissue-or bodyduct-specific positive MRI contrast agent, e.g. at least onephysiologically acceptable paramagnetic substance together with at leastone physiologically acceptable ferromagnetic or superparamagneticsubstance, preferably also together with at least one physiologicallyacceptable carrier or excipient.

The paramagnetic substance used according to the present invention maybe any one of the physiologically tolerable paramagnetic substancesalready proposed for use as MRI contrast agents. Preferably, it will bea chelate complex of a paramagnetic atom or ion, for example alanthanide or transition metal atom or ion, or a stable free radical.Conveniently, the paramagnetic species will have an atomic number of 21to 29, 42, 44 or 57 to 71. As positive MRI contrast agents, metalchelates in which the metal species is Eu, Gd, Dy, Ho, Cr, Mn or Fe areespecially preferred and Gd³⁺, Cr³⁺, Fe³⁺ and Mn²⁺ are particularlypreferred. As negative MRI contrast agents, metal chelates in which theparamagnetic metal species is Tb³⁺ or Sm³⁺ or more especially Dy³⁺ areparticularly preferred, e.g. Dy DTPA-BMA, or DyDTPA-beta-alanine-dextran(molecular weight 70000) where a blood pooling positive contrast agentis desired.

As mentioned above, the paramagnetic species is preferably present inthe form of a chelate and chelates in which the chelating atoms arenitrogen, oxygen or sulphur are particularly suitable and those in whichthe chelating moiety is a carboxylic acid or aminocarboxylic acid moietyare especially preferred.

In general, the chelating moiety in the paramagnetic substance mayconveniently be the residue of a conventional metal chelating agent.Suitable such agents are well known from the literature relating to MRIcontrast agents discussed above (see for example EP-A-71564,EP-A-130934, EP-A-186947, U.S. Pat. No. 4639365 and DE-A-3401052) aswell as from the literature relating to chelating agents for heavy metaldetoxification. The chelating moiety chosen is preferably one that isstable in vivo and is capable of forming a chelate complex with theselected paramagnetic metal species. Preferably however, the chelatingmoiety will be one as described in EP-A-186947 or the residue of anaminopoly(carboxylic acid or carboxylic acid derivative) or a saltthereof, for example one of those discussed by Schering AG inEP-A-71564, EP-A-130934 and DE-A-3401052 and by Nycomed AS in WO89/00597.

Particularly preferred as chelating moieties for the hydrophilicparamagnetic substances used in the present invention are the residuesof the following: EDTA; DTPA-BMA; DOTA; desferrioxamine; and thephysiologically acceptable salts thereof.

Where the chelating moiety in a paramagnetic substances used accordingto the present invention has a labile counterion, that counterion shouldbe a physiologically tolerable ion, for example the ion of an alkalimetal, a non-toxic amine (for example tris(hydroxymethyl)aminomethane,ethanolamine, diethanolamine and N-methylglucamine), a halogen, or anon-toxic organic or inorganic acid.

Furthermore, a paramagnetic substance used according to the inventionmay particularly conveniently comprise a paramagnetic species bound by achelating entity, itself bound to a larger mass, for example a solubleor insoluble polysaccharide macromolecule or a biomolecule which iscapable of targetting the paramagnetic species onto a specific organ ortissue within the body. In such instances, it may be advantageous tohave the chelating entity bound to the macromolecule or biomoleculethrough the agency of an intermediate or linker molecule and it may beparticularly advantageous to utilize a linker molecule which forms orcontains a biodegradable bond so that the paramagnetic centre may bereleased for excretion from the body or for uptake by particular organsor tissues.

Binding a paramagnetic metal chelate to a larger mass also has theeffect of increasing its contrast effect enhancing ability by reducingthe tumbling motion of the paramagnetic centres.

Thus for a paramagnetic negative MRI contrast agent, if uniformdistribution after i.v. administration is desired, one may convenientlyuse as the chelating moiety a hydrophilic extracellular substance, suchas DTPA or DOTA or a chelating agent as claimed in WO89/00557. However,to achieve tissue- or duct-specificity, for either positive or negativeMRI contrast agents it may instead be desired to use a paramagneticsubstance with blood-pooling properties and in this regard it may besuitable to use a chelated paramagnetic species bound to biologicallypassive or biologically tolerable macromolecules, for example proteinssuch as albumin or polysaccharides such as dextrans and cellulosederivatives, having molecular weights above the kidney threshold,preferably of about 40000 or more. Alternatively, where targetting of aparamagnetic substance onto a specific tissue is desired, theparamagnetic species may be bound to a tissue- or organ-specificbiomolecule, for example an antibody, or to chelating agents which causethe paramagnetic substance to locate at particular tissues, for examplethe meglumine salt of the chromium (III) chelate ofN-(2,6-diethylphenylcarbamoylmethyl)iminodiacetic acid (CrHIDA), theiron (III) chelate of ethylene -bis(2-hydroxy-phenylglycine)(known asFeEHPG--see Lauffer et al. Journal of Computer Assisted Tomography 9:431 (1985)), manganese chelates such as the hepatobiliary Mn²⁺ chelateof N,N'-bis (pyridoxal-5-phosphate)-ethylenediamine-N,N'-diacetic acid(Mn DPDP--see Worah et al. Society of Magnetic Resonance in Medicine,Sixth Annual Meeting, New York, 1987, Works in Progress, page 16) andthe tumor specific Mn³⁺ chelate of mesotetra (4-sulphonato-phenyl)porphine (Mn TPPS₄)(see Button et al. Society of Magnetic Resonance inMedicine, Sixth Annual Meeting, 1987, Vol. 2 Book of Abstracts, page660), lipophilic gadolinium chelates such as B-19036 (see Vittadini etal. Society of Magnetic Resonance in Medicine, Sixth Annual Meeting,1987, Vol. 1 Book of Abstracts, page 322 and also EP-A 230893) or othersubstances which are taken up by the hepatobiliary system or othertumour specific agents such as paramagnetic porphyrin derivatives.

A wide range of suitable paramagnetic substances is described in thepatents, patent applications and journal articles referred to above, thedisclosures of which are incorporated herein by reference.

Ferromagnetic or superparamagnetic substances used according to theinvention may conveniently comprise particles of a magnetic metal oralloy, for example of pure iron, or of a magnetic compound, for examplemagnetic iron oxides such as magnetite, gamma-ferrite and cobalt, nickelor manganese ferrites. These particles may be free or may be coated byor carried in or on particles of a non-magnetic matrix material. Iffree, the particles should preferably be of sufficiently small size asto be superparamagnetic, for example 5 to 50 nm. The particles of thesuperparamagnetic or ferromagnetic material may be coated or carried inor on particles of a non-magnetic matrix material, for example of apolysaccharide such as dextran, starch or a protein such as albumin andwhen targetting of the ferromagnetic or superparamagnetic substance ontoa specific tissue is desired it may be desirable to use a ferromagneticor superparamagnetic material which is bound to a tissue- ororgan-specific biomolecule (see for example Renshaw et al. MagneticResonance Imaging 4: 351-357 (1986)). The overall particle size willpreferably be less than about 100 micrometers if the particles are to beadministered orally and less than 5.0 micrometers if the particles areto be administered intravenously. For intravenous administration, themean overall particle size will preferably be in the region of 0.01 to1.5 micrometers, while for administration directly into the digestivetract (for example orally) or into the bladder, the uterus, the biliaryduct or the parotid duct, the mean overall particle size will preferablybe 0.01 to 50 micrometers, especially 0.1 to 2.0 micrometers.

Where the superparamagnetic or ferromagnetic material is provided with anon-magnetic coating or matrix, the coating or matrix material isparticularly preferably a protein or a starch or polysaccharide materialas suggested Schroder in WO83/01738 or a biotolerable polymer such as issuggested by Ugelstad et al. in WO83/03920. Biodegradable coating ormatrix materials such as suggested by Schroder are particularlypreferred for particles which are to be administered parenterally whilethe polymer matrices and coating materials of Ugelstad are particularlypreferred for oral administration.

Where the ferromagnetic or superparamagnetic material is provided with acoating or matrix, the iron content of the overall particles willpreferably be from 0.1 to 80%, more preferably at least 1%, especially 5to 70%, by weight.

As with the paramagnetic substances discussed above, a wide range offerromagnetic and superparamagnetic substances is suggested in thepatents, patent applications and journal articles mentioned above, thedisclosures of which are also incorporated herein by reference.

The positive and negative contrast agents may be administered eithertogether or separately and either parenterally or enterally, e.g.directly into a body cavity having an external evacuation duct, forexample orally or rectally or, generally by catheter, directly into thebladder or uterus. Generally, the positive and negative MRI contrastagents will be administered into the same body duct, organ or cavity,for example both may be administered intravenously; nevertheless incertain circumstances it may be desirable to select differentadministration routes for the positive and negative contrast agents andto image body sites where the separately administered agents approach ormerge. Consequently, the contrast media administered to the patient areconveniently either unitary compositions containing both positive andnegative contrast agents or are separate compositions one containing atleast one negative contrast agent and another containing at least onepositive contrast agent and in a further aspect the present inventionalso provides a MRI contrast medium kit comprising a first containercontaining a positive MRI contrast medium (e.g. a paramagneticsubstance, together with at least one physiologically acceptable carrieror excipient, for example water for injections) in an administrationform adapted for body tissue- or body duct-specific contrast enhancementand a second container separately containing a negative MRI contrastmedium (e.g. a superparamagnetic or ferromagnetic substance togetherwith at least one physiologically acceptable carrier or excipient) in anadministration form adapted for body duct- or body tissue-specificcontrast enhancement. Particularly conveniently the positive andnegative media in the kit of the invention are adapted foradministration into different body tissues, ducts or cavities.

The kit of the invention may be used for the separate administration ofthe positive and negative contrast agents or the positive and negativecontrast agents from a kit may be mixed and administered together.

Thus, for example, the kit of the invention might conveniently containtwo intravenous contrast agents, for example a dispersion ofintravenously administrable ferromagnetic or superparamagnetic particlesand an intravenously administrable solution of a soluble dextran-boundgadolinium chelate, or the kit might contain an orally administrablecontrast agent and an intravenous contrast agent, for example adispersion or suspension of magnetic particles for administration intothe gastrointestinal tract and an intravenous solution containing aparamagnetic substance which will target onto the pancreas.Alternatively, to obtain systemic contrast enhancement, an orallyadministrable non-absorbable negative contrast agent and an orallyadministrable absorble paramagnetic substance might be used, eitherformulated together as a single composition or formulated separately,for example for packaging together as a kit according to the invention.

The method of the present invention appears to be particularly promisinginsofar as the imaging of the hepatobiliary system is concerned.

Thus it has been shown in animal experiments that intravenousadministration of superparamagnetic ferrite particles embedded in starchmatrix particles and of a hepatobiliary paramagnetic contrast agent(CrHIDA) results in the production of greatly improved magneticresonance images of the hepatobiliary system. With the superparamagneticparticles alone, a negative contrast effect was observed in the liverbut the biliary system could not be observed. With CrHIDA at doses atwhich it has a positive contrast effect, the imaging of the gall bladderwas enhanced but it was not possible to observe the bile ducts in theliver; however on administration of both the positive and negative MRIcontrast agents, the biliary tree, including very tiny bile ducts in theliver, could be observed. Thus the combination of positive and negativetissue specific MRI contrast agents allowed in vivo imaging of theanatomical structure of the liver to an extent that was not possibleusing either of the agents separately.

The preferred dosages of the positive and negative contrast agents usedaccording to the present invention will vary over a wide range and thechosen dosage will depend upon such factors as the administration route,the nature of the subject, the biodistribution, pharmacokinetics andchemical nature of the contrast agents, and the magnetic field strengthand pulse sequence used in the imaging routine. In general, the dosageswill be similar to the dosages suggested for the paramagnetic,ferromagnetic and superparamagnetic contrast agents used alone.Conveniently, the dosage for a paramagnetic positive or negativecontrast agent will be in the range of 0.001 to 10 mmol/kg bodyweight,especially 0.01-1 mmol/kg bodyweight, while the dosage for aferromagnetic or, preferably, superparamagnetic substance will be in therange 0.0001 to 5 mmol/kg bodyweight, preferably 0.001-1 mmol Fe/kgbodyweight.

The contrast agent compositions used according to the present inventionmay of course contain other components besides the paramagnetic,ferromagnetic or superparamagnetic substances and in this regardparticular mention may be made of viscosity modifiers, flavourings, pHadjusting agents, osmolality regulators, stabilizers, antioxidants,buffers and emulsifying or dispersing agents as well as otherconventional pharmaceutical or veterinary formulation aids.

The contrast media may be formulated in conventional pharmaceuticaladministration forms, such as tablets, capsules, powders, solutions,suspensions, dispersions, syrups, suppositories, etc.; however,solutions, suspensions and dispersions in physiologically acceptablecarrier media, for example water for injections or physiological saline,will generally be preferred.

Where the medium is formulated for parenteral administration, thecarrier medium incorporating the magnetic substances will preferably beisotonic or somewhat hypertonic.

For magnetic resonance diagnostic examination, the paramagneticsubstance, if in solution, suspension or dispersion form, will generallycontain the paramagnetic metal species at a concentration in the range 1micromole to 1.5 mole per litre, preferably 0.01 to 1000 millimole.

The composition may however be supplied in a more concentrated form fordilution prior to administration. The ferromagnetic or superparamagneticsubstance will generally be presented in the form of a suspension ordispersion at a concentration of 0.001 to 10 mol/liter iron. Again, itmay be supplied in a more concentrated form for dilution prior toadministration.

Where the method of the invention involves parenteral administration ofa particle-containing composition, it may be desirable to sonicate thecomposition before administration to ensure uniform dispersion of theparticles; this may however be unnecessary for many superparamagneticdispersions.

The present invention will now be illustrated further by reference tothe following non-limiting Examples in which ratios, percentages andparts referred to are by weight unless otherwise indicated:

EXAMPLE 1 Intravenous contrast agent for visualization of the liver andbiliary system

    ______________________________________                                        Composition:                                                                  ______________________________________                                        Meglumine salt of the chromium (III) chelate of N-                                                        1333   mg                                         (2,6-diethylphenyl-carbamoylmethyl)iminodiacetic                              acid (i.e. CrHIDA)                                                            Starch-magnetite particles  200    mg                                         Aqua purificata ad          20     ml                                         ______________________________________                                    

The meglumine salt of the chromium (III) chelate ofN-(2,6-diethyl-phenylcarbamoylmethyl)-iminodiacetic acid was prepared inaccordance with Example 12 of EP-A-165728 and dissolved in distilledwater. Starch-magnetite particles (0.1-0.7 micrometer diametercontaining 70% by weight iron) were prepared by the Schroder method andadded to the red solution of the chromium chelate. The mixture wassonicated for 5 minutes followed by sterilization. The product wasfilled into a 20 ml vial. Each vial contained 0.075 mmol Cr/ml and 20 mgstarch-magnetite particles/ml.

EXAMPLE 2 Parenteral contrast agent for visualization of liver andspleen together with the vascular system in these organs

    ______________________________________                                        Composition:                                                                  ______________________________________                                        Gadolinium(III)DTPA-beta-alanine-dextran                                                                 78.6   mg                                          Dextran-magnetite-complex  50     mg                                          Saline solution (0.9% sodium chloride) ad                                                                10     ml                                          ______________________________________                                    

Gadolinium(III)DTPA-beta-alanine-dextran, prepared in accordance withExample 3 below, was dissolved in 0.9% sodium chloride solution.Superparamagnetic dextran-magnetite (from Meito Sangyo, Japan) wasadded. The mixture was sonicated for 5 minutes and sterilized. Theproduct was filled into a 10 ml vial. The vial contained 0.05 mmol Gd/mland 5 mg dextran-magnetite complex/ml.

EXAMPLE 3 GdDTPA-beta-alanine-dextran (Molecular Weight 70,000)

To a solution of 15.9 g of dextran (molecular weight 70,000, availablefrom Sigma Chemicals) in 650 ml of dry dimethyl sulphoxide (DMSO) wasadded 20.3 g of fluorenylmethyloxycarbonyl-beta-alanine, 13.7 g ofN-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide and 968 mg of4-pyrrolidino-pyridine dissolved in 350 ml of dry DMSO. The reactionmixture was stirred at ambient temperature for 18 hours and 43.1 g ofpiperidine was added. After 70 minutes, 7.3 ml of concentratedhydrochloric acid was added dropwise, and cooling on an ice/water bathand dropwise addition of 1.7 l of an ether/chloroform mixture (7:3 w/w)yielded a yellow oil. After decantation, the oil was dissolved indistilled water and the pH was adjusted to 4. Sodium chloride was addeduntil the salt concentration was 0.9% in 1400 ml of solution, and theproduct was dialyzed against 0.9% sodium chloride in water at pH 4 in ahollow fibre cartridge (Amicon HP 10-20) for 24 hours. The solution wasthen concentrated using the same equipment against distilled water to avolume of 1150 ml, the pH was adjusted to 9 with N-methylmorpholine and29.18 g of DTPA-bis-anhydride was added while the pH was kept at 8 usingthe same base. When the solution became clear, the reaction mixture wasstirred for 2 hours, 43.78 g of citric acid dissolved in 47.4 ml of 10 NNaOH was added, and the pH was adjusted to 6.0 with concentratedhydrochloric acid. 30.37 g of gadolinium chloride hexahydrate dissolvedin 200 ml of distilled water were added quickly and the pH was adjustedto 5.5 using 10 NaOH. The solution was dialyzed against distilled wateruntil the relaxation time T₁ (determined using a NMR Proton SpinAnalyzer, RADX Corporation, Houston, Tex., USA, at 10 MHz and 37° C.)was above 2000 ms. Lyophilization of the solution yielded 15.3 g of alight yellow coloured powder.

ANALYSIS Elemental analysis:

Gd 4.6%; N 2.15%; Na 0.16%; Cl less than 0.01%.

Free Gd (xylene orange titration), DTPA, GdDTPA, citric acid, or DMSO(HPLC): less than 0.01% (The percentages in the analysis results are byweight).

EXAMPLE 4 Kit of two different contrast agents--suspension for oraladministration to obtain a negative contrast effect in thegastrointestinal tract and solution for intravenous administration toobtain positive contrast enhancement of the hepatobiliary system

    ______________________________________                                        Suspension for oral administration:                                           ______________________________________                                        Magnetic particles      1.0    g                                              Hydroxyethyl cellulose  10.0   g                                              Methyl parahydroxybenzoate                                                                            0.8    g                                              Propyl parahydroxybenzoate                                                                            0.2    g                                              Ethanol                 10.0   g                                              Saccharin sodium        1.0    g                                              Orange essence          0.3    g                                              Apricot essence         0.7    g                                              Water ad                1000   ml                                             ______________________________________                                    

Hydroxyethyl cellulose was dispersed in water with stirring for 2 hours.Saccharin sodium and a solution of the essences, methyl and propylparahydroxybenzoate in ethanol were slowly added. Magnetic particles (3micrometer in size and containing 19.4% by weight iron) were prepared inaccordance with WO 83/01738 (particle type 1) and dispersed in thesolution under vigorous stirring. The suspension was filled into a 1000ml bottle. The suspension contained 194 mg iron.

    ______________________________________                                        B-19036                  6 g                                                  Aqua Purificata ad      10 ml                                                 ______________________________________                                    

The gadolinium(III) salt is dissolved in distilled water. The solutionis filled into a 10 ml vial and heat sterilized.

EXAMPLE 5 Intravenous paramagnetic contrast agent

An injection solution is prepared containing:

    ______________________________________                                        CrHIDA                  6.67   g                                              Water for injections ad 100    ml                                             ______________________________________                                    

The chromium salt is dissolved in the water for injections, sterilefiltered through 0.22 micrometer millipore filter and filled into 10 mlvials under aseptic conditions.

EXAMPLE 6 Intravenous superparamagnetic contrast agent

An injectable dispersion is prepared which contains:

    ______________________________________                                        Magnetic particles      30    mg                                              Water for injections ad 10    ml                                              ______________________________________                                    

The magnetic particles, which are prepared according to Example 7 below,are dispersed in the water for injections and filled into 10 ml vialsunder aseptic conditions. A kit is made up containing vials of theparamagnetic agent of Example 5 and vials of the superparamagnetic agentof Example 6. The superparamagnetic agent is sonicated beforeadministration to ensure complete dispersion of the magnetic particles.

EXAMPLE 7 Magnetic particles

Using the Schroder method, ferrite particles of a mean particle size of10 nm are embedded in starch to produce ferrite/starch particles havinga mean particle size of 0.7 micrometer (the majority of the particlesbeing in the size range 0.3 to 1.1 micrometers) and an Fe content ofabout 60% by weight.

EXAMPLE 8 Imaging of the Hepatobiliary system in dogs

Beagle dogs weighing 10 to 12 kg were examined by magnetic resonanceimaging under iv pentothal anaesthesia. In eight dogs, examinations wereperformed over four days before and after i.v. injection of 4 to 8mgFe/kg bodyweight of superparamagnetic iron oxide (the composition ofExample 6). Examinations were made directly, every hour for four hours,every two to three hours up to twelve hours and around 24, 48 and 72hours after injection of the contrast agent. In three other dogs,examinations were performed before and after i.v. injection of 0.1 to0.2 mmol/kg bodyweight of CrHIDA (the composition of Example 5). Fourfurther dogs were examined before and after i.v. injection of 2 to 4mgFe/kg bodyweight of superparamagnetic iron oxide followed after about30 minutes by 0.2 to 0.4 mmol/kg bodyweight of CrHIDA i.v. Examinationsafter CrHIDA were made every 10 minutes for 60 minutes.

Examination was by magnetic resonance imaging using a superconductivesystem (Siemens Magnetom) operating at 0.5 tesla. After CrHIDAadministration, examinations were made in the head coil and in thetransverse and frontal projections with 7 or 20 mm slice thicknesses ina multislice mode with TR of 0.25 sec and TE of 22 and 35 msec. Agradient echo sequence (FLASH) with an 80° flip angle, TR of 140 ms andTE of 14 ms was used in one examination. In the case of theadministration of the superparamagnetic contrast agent alone, multislicetransverse images were taken with TR 500, TE 22 and TR 1500, TE 35/70msec. For T₁ measurements, 6 TR of 100, 300, 500, 700, 1500 and 3000 mswith TE 20 ms and for T₂ measurements four TE of 30, 60, 94 and 134 mswith TR 1500 ms were used.

In three of the dogs to which the positive and negative contrast agentswere administered, 1.0 unit/kg bodyweight of cholecystokinin were givenintravenously 60 minutes after administration of the paramagneticcontrast agent immediately followed by examinations in the transverseand frontal projections.

Blood, urine and liver function tests were performed two days after theinvestigations.

Before contrast administration, the bile ducts were not discernible,while the gall bladder in some cases could be identified because of itsslightly higher signal intensity than that of the liver. In the dogsexamined after administration of only CrHIDA, the bile ducts were notvisible either, although a higher signal intensity was present in thegall bladder after 15 to 30 minutes. The signal intensity of the liverparenchyma or of the muscle did not change significantly after injectionof CrHIDA.

After injection of the superparamagnetic contrast agent at a dosage of2.0 mgFe/kg bodyweight, a pronounced lowering of the signal intensity ofthe liver was achieved and further reduction of the signal close to thebackground noise level was present after 4.0 mgFe/kg was noted. Themaximum effect was noted after about 30 minutes and thereafter persistedunchanged throughout the measurement. There was no change of the signalintensity of the muscle.

The wider bile ducts were discernible after administration of thesuperparamagnetic contrast agent as a result of the lowering of thesignal intensity from the liver. The gall bladder was also readilydiscernible. However, after administration of the superparamagneticcontrast agent followed by administration of the paramagnetic contrastagent, the bile ducts exhibited a higher signal intensity and even verytiny bile ducts were visible in the transverse and frontal images. Anincreased signal intensity from the gall bladder was also encountered 15to 30 minutes after contrast agent administration. After administrationof the superparamagnetic and paramagnetic contrast agents and aftercholecystokinin injection, the gall bladder was moderately contractedand visualization of the choledocus duct was achieved as well ascontrast filling of the duodenum.

The blood, urine and liver function texts were found to be normal duringthe experiments.

FIGS. 1 to 3 of the accompanying drawings illustrate the observedenhancement in liver image contrast.

FIGS. 1a, 1b and 1c are transverse images of the liver before contrastagent administration (FIG. 1a), after administration of the negativecontrast agent (FIG. 1b) and after administration of both negative andpositive contrast agents (FIG. 1c). Small bile ducts not discernible inFIGS. 1a and 1b become visible in FIG. 1c.

FIGS. 2a, 2b and 2c are similarly transverse images of the liver beforeand after contrast agent administration and FIGS. 3a, 3b and 3c arefrontal images of the liver before and after contrast agentadministration.

For FIGS. 1 to 3 the contrast agent dosages are respectively

    ______________________________________                                                  Negative Agent                                                                          Positive Agent                                            ______________________________________                                        FIG. 1 (a, b & c)                                                                         2.0 mg Fe/kg                                                                              0.2 mmol/kg Cr HIDA                                   FIG. 2 (a, b & c)                                                                         4.0 mg Fe/kg                                                                              0.4 mmol/kg Cr HIDA                                   FIG. 3 (a, b & c)                                                                         4.0 mg Fe/kg                                                                              0.4 mmol/kg Cr HIDA                                   ______________________________________                                    

We claim:
 1. A magnetic resonance imaging contrast medium comprising atleast one physiologically acceptable body tissue- or body duct-specificnegative MRI contrast agent together with at least one physiologicallyacceptable body tissue- or body duct-specific positive MRI contrastagent.
 2. A medium as claimed in claim 1 adapted for administration intothe cardiovascular system and wherein one or both of said positive andnegative agents is a blood-pool agent.
 3. A medium as claimed in claim 1comprising at least one physiologically acceptable paramagneticsubstance together with at least one physiologically acceptableferromagnetic or superparamagnetic substance.
 4. A medium as claimed inclaim 1 wherein said positive agent comprises a chelate of Gd³ +, Cr³ +,Fe³ + or Mn² +.
 5. A medium as claimed in claim 1 wherein said negativeagent comprises a chelate of Dy³ +, Tb³ + or Sm³ +.
 6. A medium asclaimed in claim 1 where said negative agent comprises superparamagneticparticles carried by particles of physiologically acceptable matrixmaterial.
 7. A medium as claimed in claim 1 adapted for administrationinto the cardiovascular system and comprising superparamagneticparticles carried by particles by a physiologically acceptable matrixmaterial together with a positive contrast agent selected from CrHIDA,FeEHPG and macromolecule-bound chelated paramagnetic metal specieshaving a molecular a weight of at least 40,000.
 8. A MRI contrast mediumkit comprising a first container containing a negative MRI contrastmedium in an administration form adapted for body tissue- or bodyduct-specific contrast enhancement and a second container separatelycontaining a positive MRI contrast medium in an administration formadapted for body tissue- or body duct-specific contrast enhancement. 9.A kit as claimed in claim 8 wherein said positive and negative media arein forms adapted for administration into different body tissues, ductsor cavities.
 10. A method of generating images of the human or non-humananimal body, which method comprises administering to said body adiagnostically effective amount of a body tissue- or body duct-specificMRI contrast agent and of a body tissue- or body duct-specific positiveMRI contrast agent and generating a magnetic resonance image of a partof said body containing said negative and positive agents orparamagnetic, ferromagnetic, or superparamagnetic biodegradationproducts thereof.
 11. A method as claimed in claim 10 wherein one orboth of said positive and negative agents is a blood-pool agent and isadministered into the cardiovascular system of said body.
 12. A methodas claimed in claim 10 wherein said positive and negative agents areadministered together.
 13. A method as claimed in claim 10 wherein saidpositive and negative agents are administered via different routeswhereby to approach or merge at a selected body site and wherein saidimage is generated of said selected body site.
 14. A method as claimedin claim 10 wherein as said negative agent is administered a particulateferromagnetic or superparamagnetic material.
 15. A method as claimed inclaim 10 wherein said negative agent is administered a physiologicallyacceptable chelate of Dy³ +, Tb³ + or Sm³ +.
 16. A method as claimed inclaim 10 wherein as said positive contrast agent is administered aphysiologically acceptable chelate of Gd³ +, Cr³ +, Fe³ + or Mn² +. 17.A method as claimed in claim 10 wherein a physiologically acceptableparamagnetic substance is administered as said MRI contrast agent.